1. Field of the Invention
The present invention generally relates to microfluidic devices and methods for use, and more particularly, to high-throughput microfluidic devices and methods for on-chip complex biological and/or chemical sample separation and/or detection of separated components.
2. Discussion of Related Art
Cancer is a leading cause of death in the U.S. and throughout the world, with over 1,000,000 new cases being diagnosed every year in the U.S. alone. Hundreds of genes are involved in the development of diseases such as cancer. An accumulation of mutations, which result in loss- or over-expression of genes that control cell growth and proliferation, ultimately lead to the onset of the disease. To fully elucidate the molecular mechanisms and pathways that govern cancer initiation and progression, the information generated at the DNA and mRNA level must be complemented with a complete and detailed panorama of protein expression levels and their post-translational modifications. The characterization of the proteomic complement of a specific genome will enable us to understand the basic processes that distinguish healthy versus diseased states.
The analysis of proteomic samples is very difficult, however, for a number of reasons. First, the sample is either available in a limited amount (e.g., a few thousands of cells to start with), or the final subfractions submitted to analysis are extremely small (e.g., 1-10 μl at the nM/pM concentration level). Second, the sample itself is typically complex (5,000-10,000 different proteins may be expressed in a mammalian cell at any given time). Third, the dynamic range of the proteins present is typically very large (1:106) and/or their presence easily obscured by other more highly abundant components.
In order to contend with the overwhelming numbers of mixture components, complex samples are typically separated (i.e., fractionated) into smaller components prior to analysis. Two common types of separation techniques include: “gel-based” (e.g., 2D Polyacrylamide Gel Electrophoresis “2D-PAGE”); and “liquid chromatography (LC)-based” (e.g., 2D Strong Cation Exchange “SCX” and High Performance Liquid Chromatography “HPLC”) approaches. Comparatively, 2D-PAGE is a relatively awkward and time consuming separation technique that utilizes a gel to separate proteins according to isoelectric point, or charge, in a first dimension, and according to molecular weight, or size, in a second dimension. LC techniques utilize a stationary phase and a mobile phase to separate sample components based on properties, such as hydrophobicity, charge, and size. LC operates such that the sample to be separated is initially loaded at the head of a separation channel packed with a stationary phase, and then forced through the channel with the aid of a mobile phase under high pressure. The sample components are separated according to their differential interaction with the packing material (the stationary phase) in the channel. In 2D-LC, for example, the sample is separated according to charge in the first dimension and according to hydrophobicity in the second dimension. Several notable advantages of 2D-LC over the more complicated 2D-PAGE process include: easier automation, no gel handling, better separation power, and simpler coupling to other techniques, such as mass spectrometry.
Mass Spectrometry (MS) is a detection technique commonly used following the above mentioned separation techniques. MS is used to detect sample components with a higher degree of detail by generating a mass spectrum representing masses of individual components. In addition, MS is highly suited for proteomic investigations for a number of reasons: 1) it generates results with a level of confidence that is comparable to amino acid sequencing of electrophoretically separated proteins; 2) it is more tolerant towards low molecular contaminants; and 3) it is faster, simpler, and more sensitive than traditional sequencing protocols. As a result, MS has evolved into the detection tool of choice for proteomic applications (refs 1-5) as it offers the combined benefits of specificity, sensitivity, resolving power, and capability to deliver high quality structural information. In many cases, routine MS analysis can be performed from protein quantities as low as 10-100 fmol; and state-of-the-art MS is demonstrating results at the amol level.
Other recent improvements in MS detection sensitivity and analysis speed have launched this technique into an effective strategy for the detection of novel disease biomarkers and protein co-expression patterns. Current approaches for biomarker discovery and/or screening involve techniques such as DNA microarray technology, immunohistochemical staining, protein chips, and multidimensional separations followed by MS detection. These approaches, however, are accompanied by numerous limitations that include: insufficient quantitative correlation between gene and protein expression level; complexity and lengthiness for analytical approaches that attempt comprehensive qualitative/quantitative proteomic profiling; and lack of sufficient sensitivity, specificity and reproducibility.
Mass spectrometry generally operates by initially ionizing the sample within an ionization source, separating the ions of differing masses, and recording their relative abundance with a mass spectrometer. In particular, two commonly used MS ionization techniques include: Electrospray ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI). ESI operates by generating gas-phase ions (or “spray”) from a liquid phase solution. According to this technique, an analyte solution is pumped through a very small charged capillary such that the liquid exits the capillary in the form of an aerosol or mist. After a series of subsequent charge-based interactions, the aerosol droplets eventually reach the mass spectrometer in the form of lone ions for detection. MALDI, on the other hand, is an ionization technique that mixes a matrix solution with the sample (or analyte) to produce a homogeneous co-crystallized analyte-matrix solution. Specifically, the role of the matrix is to absorb the energy of a laser beam and transfer it to the more fragile analyte molecules. Advantages of MALDI-MS include: amenability to high-throughput, ease of operation, and applicability for fast peptide mapping. Recently, MS/MS sequencing techniques that enable unambiguous peptide identifications have been developed for MALDI-MS as well. However, some challenges toward this end include developing simple, compact, and low cost devices with parallel processing capabilities that would enable high-throughput investigations.
Interestingly, miniaturization is emerging as a significant trend in analytical and biological instrumentation (refs 6-8). One unique benefit of miniaturization is that a variety of novel analytical configurations only become possible if they are developed in a microfabricated format. For example, high-speed capillary electrophoresis (CE) can be performed on a microchip device in as fast as 0.8 ms using a separation channel of only 200 μm in length (ref 9). Moreover, microchips can handle as little as 1-5 microliters (μl) of sample, analyze volumes as low as 1 pl-1 nl, and perform sample injection, labeling, and detection within millisecond time-frames (ref 9). Additionally, the micro-domain environment enables the emergence of unique physical events. Particularly, as the size of a device decreases, the surface-to-volume ratio increases. As a result, surface driven phenomena begin to dominate in the micro-scale world. Electroosmotic flow (EOF), which can be effectively generated only in capillaries with dimensions in the micrometer (um) domain, represents a relevant example.
Microfluidic devices function according to well-established principles and are characterized by several features that distinguish them from large-scale instrumentation. First, the miniature format of these devices enables the manipulation of extremely small sample amounts and short analysis times, resulting in significantly reduced analysis costs. Second, the ability to perform precise and accurate sample handling operations with microfluidic devices advantageously enables process control and automation, and the generation of reliable and high-quality data. Finally, microfabrication enables large-scale integration, multiplexing, and consequently high-throughput analysis. In addition, microfluidic devices may be fabricated from several different materials that include glass, quartz, polymeric and silicon substrates (refs 6,8), and may comprise a variety of elements that perform operations, such as pumping, dispensing, clean up, mixing, separation, chemical alterations, and detection (ref 7).
Despite the advantages of microfluidic devices, very few have been successfully combined with MS analysis (refs 10-30). In addition, because MS/MS capabilities were only available until recently with ESI-MS, the microchips that have been developed have been primarily interfaced with ESI. Such microchip-ESI interfaces typically allow ESI to be generated either: directly from the chip surface (refs 10-12); from capillary emitters inserted in the chip (13-18); or from microfabricated emitters (refs 19-22). Liquid sheath, liquid junction and nano-ESI sources have been implemented (refs 17-23). However, the bottleneck of all these applications goes back to the sequential nature of traditional ESI-MS detection. Conversely, microfluidic technologies that enable high-throughput MALDI-MS detection remain in a very early stage of development. These technologies typically make use of a piezo-actuated flow-through dispenser (refs 24,25) or a centrifugal CD (refs 31,32). For example, microfabricated piezo-actuated flow-through dispensers can be used to deposit samples on a nanovial target plate and to interface capillary liquid chromatography to MALDI/TOF (refs 24,25). Alternatively, the centrifugal CD enables sample loading at the center of the disc, centrifugal transport through reaction chambers to the outer edge of the disc, and sample collection into small spots on the edge of the CD for MALDI-MS detection (refs 31,32). In addition, array-based technologies, such as the surface enhanced laser desorption ionization (SELDI) approach, have been shown to enable MALDI-MS detection from functionalized protein chips (ref 38).
Although the above microfluidic devices have demonstrated noteworthy sensitivity, throughput, and flexibility, they do not demonstrate the full benefits of microfluidic platforms combined with MS detection for a number of reasons. First, as mentioned, the main limitation of ESI-MS detection in the context of microfabricated analysis platforms relates to the traditionally sequential nature of the technique. This attribute inherently forfeits some of the greatest advantages provided by miniaturization, i.e., large-scale integration, multiplexing, and high-throughput analysis. Also, the fabrication of microfabricated ESI spray emitters is difficult, and their integration with microfluidic chips that perform complex sample processing steps has not been demonstrated. Second, the fabrication of piezo-actuated micro-dispensers that enable sample collection from multiplexed microfluidic chips for MALDI-MS detection is not simple, and their integration within sample processing chips has not been demonstrated either. Moreover, the dispensing process would deposit the sample onto a different chip than the one used for analysis, consequently increasing labor and costs. Third, the centrifugal CD does not enable the integration of a separation step prior to MS analysis since sample collection occurs in a single spot. Fourth, intense scrutiny of the SELDI chip approach has demonstrated that the technique is neither sufficiently reproducible, nor sensitive enough to detect low abundance signature biomarker components (ref 34). Also, the technique does not enable dynamic processing of biological samples as required for protein analysis.
Overall, microfluidic chips that enable complex sample processing followed by high throughput MS detection do not exist at the present time. For example, existing microfluidic devices may perform on-chip separation, but are subsequently connected to external pumping systems and autosamplers for MS detection. Such techniques do not provide sufficient throughput and cost-effective analysis. Therefore, what is needed is a small, microfluidic device comprising all the components necessary to perform complex sample separation as well as enable on-chip MS detection. Additionally, what is needed is a microfluidic device for complex sample processing that provides high sensitivity and throughput, requires only small amounts of complex samples, and minimizes overall analytical costs.